Essential roles of three enhancer sites in σ54-dependent transcription by the nitric oxide sensing regulatory protein NorR

The bacterial activator protein NorR binds to enhancer-like elements, upstream of the promoter site, and activates sigma(54)-dependent transcription of genes that encode nitric oxide detoxifying enzymes (NorVW), in response to NO stress. Unique to the norVW promoter in Escherichia coli is the presence of three enhancer sites associated with a binding site for sigma(54)-RNA polymerase. Here we show that all three sites are required for NorR-dependent catalysis of open complex formation by sigma(54)-RNAP holoenzyme (Esigma(54)). We demonstrate that this is essentially due to the need for all three enhancers for maximal ATPase activity of NorR, energy from which is used to remodel the closed Esigma(54) complex and allow melting of the promoter DNA. We also find that site-specific DNA binding per se promotes oligomerisation but the DNA flanking the three sites is needed to further stabilise the functional higher order oligomer of NorR at the enhancers.